RNA–protein interactions promote asymmetric sorting of the ASH1 mRNA ribonucleoprotein complex

The asymmetric distribution of Myo4p and She3p in anaphase cells is dependent on She2p mRNA-binding activity, but formation of the Myo4p/She3p complex is independent of She2p RNA-binding activity. (A) Representative images for the intracellular distribution of Myo4p-myc₁₃ in cells devoid of She2p, expressing wild-type She2p, She2p mutant N36S, and She2p mutant R63K. Strain YLM1309 was transformed with plasmid YCplac111 (vector), pRL453 (YCplac111-She2p-wt), pRL462 (YCplac111-She2p-N36S), or pRL463 (YCplac111-She2p-R63K). Transformants were grown in synthetic media minus leucine; subsequently, the cells were fixed and processed for immunofluorescence using anti-myc. Then 100 anaphase cells with signal corresponding to Myo4p were counted; below each panel, the percentage of anaphase cells with asymmetrically sorted Myo4p is indicated. (B) Representative images for the intracellular distribution of She3p-myc₁₃ in cells devoid of She2p, expressing wild-type She2p, She2p mutant N36S, or She2p mutant R63K. Strain YLM1368 was transformed and processed for immunofluorescence identically to strain YLM1309 described above. Then 100 anaphase cells with signal corresponding to She3p were counted; below each panel, the percentage of anaphase cells with asymmetrically sorted She3p is indicated. (C) The Myo4p/She3p complex can form in the absence of She2p RNA-binding activity. Strain YLM921 was transformed with plasmids (lane 1) pRL460 (YCplac22-She3p-myc₆)/pRL453 (YCplac111-She2p-wt). Yeast strain YLM1320 was transformed with the following combinations of plasmids: (lane 2) vector (YCplac22)/pRL453, (lane 3) pRL460/pRL453, (lane 4) pRL460/YCplac111 (vector), (lane 5) pRL460/pRL462 (YCplac111-She2p-N36S), or (lane 6) pRL460/pRL463 (YCplac111-She2p-R63K). Transformants were grown in synthetic media minus leucine and tryptophan. Cells were harvested, lysates were prepared, and an aliquot of the total lysate was saved for Western blotting. She3p-myc₆ was immunoprecipitated from the remaining portion of each lysate with anti-myc. The yeast lysates (total) and immunoprecipitates were analyzed by Western blot for the presence of She3p-myc₆ and Myo4p-3HA.